Alessandra Valentini

Valproic acid induces apoptosis, p16INK4A upregulation and sensitization to chemotherapy in human melanoma cells.

It is known that melanoma develops as a consequence of multifactorial alterations. To date several studies indicate the effective implication of p16 as a tumor suppressor gene with a major role in either the development or progression of human melanoma. Deregulation of melanoma cell growth has been widely associated with mutations in the p16-cyclin D/cdk4-pRb pathway. Recently anticancer therapies are focused on restoration of p16 CDK inhibitory function and other proteins unregulated in melanoma cell cycle pathway (e.g., c-myc, p27). A combined strategy for restoration of normal homeostasis in the melanoma skin with targeted delivery of apoptosis-inducing agents does not seems to be far obtain. New class of antitumoral agents are emerging: histone deacetylase (HDAC) inhibitors have attracted much interest because of their ability to arrest cell growth, induce cell differentiation, and in some cases, induce apoptosis of cancer cells. Recently, attention has been focused on the ability of HDAC inhibitors to induce perturbation in cell cycle regulatory protein (e.g., p21CIP1) and down-regulation of survival signalling pathway. In the present study, we have examined the effect of valproic acid (VPA) on M14 human melanoma cell line. Here we observed that VPA induces cell cycle arrest and apoptosis sensitising melanoma cells to cis-platin and etoposide treatment. IC50 dose (2,99 mM) of VPA was able to induce G1 arrest (up to 75 %) in association with up-regulation of p16, p21 and cyclin-D1 related to Rb ipo-phosphorilation. In addition VPA activated apoptosis (50 %) in M14 cells, when given alone or in combination with antitumoral agents. The ability of valproic acid to re-established the G1 pathway in melanoma cells suggests a potential application of VPA in melanoma therapeutic protocols.

Genetic polymorphisms of glutathione S-transferases GSTM1, GSTT1, GSTP1 and GSTA1 as risk factors for schizophrenia.

Oxidative damage is thought to play a role in the predisposition to schizophrenia. We determined if the polymorphisms of the GSTP1, GSTM1, GSTT1 and GSTA1 genes, which affect the activity of these enzymes against oxidative stress, have a role as susceptibility genes for schizophrenia, analyzing 138 schizophrenic patients and 133 healthy controls. We found that the combination of the absence of GSTM1 gene with the of the GSTM1 gene with the polymorphism GSTA1*B/*B, and the presence of the GSTT1 gene, represents a risk factor for schizophrenia, indicating that the combination of different GST polymorphisms has a role in the predisposition to schizophrenia, probably affecting the capacity of the cell to detoxify the oxidized metabolites of catecholamines.

DNA binding and cytotoxicity of fluorescent curcumin-based Zn(II) complexes

Two new heteroleptic pentacoordinated Zn(II) complexes (1 and 2) containing 4,4′-disubstituted 2,2′-bipyridines as the main ligand and curcumin (curc) as an ancillary ligand have been synthesized, spectroscopically and structurally characterized, and tested in vitro towards different human cancer cell lines. While the nitrogen ligands are almost inactive, Zn(II) curc derivatives 1 and 2 show promising and selective anticancer properties. In particular the curc Zn(II) complex 1 shows the strongest growth inhibition in all cell lines, being even more effective than the pure curc in the LAN-5 neuroblastoma cell line. Furthermore, the curc moiety makes the complexes 1 and 2 fluorescent, a feature enabling investigation of their interaction with DNA through a new optical method previously tested with the reference fluorescent intercalator ethidium bromide. This analysis demonstrates that the interaction mode of curc, 1 and 2 with DNA in the double helix favors their alignment perpendicular to the DNA axis, suggesting a partial inter-base intercalation of these Zn(II) complexes.

Effects of dutasteride on the expression of genes related to androgen metabolism and related pathway in human prostate cancer cell lines

SummaryAndrogens play an important role in controlling the growth of the normal prostate gland and in the pathogenesis of benign prostate hyperplasia, and prostate cancer. Although testosterone is the main androgen secreted from the testes, dihydrotestosterone (DHT), a more potent androgen converted from testosterone by 5α-reductase isozymes, type I and II, is the major androgen in the prostate cells. The aim of this study is to investigate the cellular and molecular effects of dutasteride, a potent inhibitor of 5α-reductase type I and type II, in androgen-responsive (LNCaP) and androgen-unresponsive (DU145) human prostate cancer(PCa) cell lines. The expression pattern of 190 genes, selected on the basis of their proved or potential role in prostate cancerogenesis related to androgen signalling, were analysed using a low density home-made oligoarray (AndroChip 2). Our results show that dutasteride reduces cell viability and cell proliferation in both cell lines tested. AndroChip 2 gene signature identified in LNCaP a total of 11 genes differentially expressed (FC ≥ ±1.5). Eight of these genes, were overexpressed and three were underexpressed. Overexpressed genes included genes encoding for proteins involved in biosynthesis and metabolism of androgen (HSD17B1;HSD17B3;CYP11B2), androgen receptor and androgen receptor co-regulators (AR;CCND1), and signal transduction(ERBB2; V-CAM; SOS1) whereas, underexpressed genes (KLK3; KLK2; DHCR24) were androgen-regulated genes (ARGs). No differentially expressed genes were scored in DU145. Microarray data were confirmed by quantitative real-time PCR assay (QRT-PCR). These data offer a selective genomic signature for dutasteride treatment in prostate epithelial cells and provide important insights in prostate cancer pathophysiology.

Myc down-regulation induces apoptosis in M14 melanoma cells by increasing p27 kip1 levels

In recent years, increasing evidence indicated the importance of a deregulated c-myc gene in the melanoma pathogenesis. We have previously demonstrated that treatment of melanoma cells with c-myc antisense oligodeoxynucleotides can inhibit cell proliferation and activate apoptosis. To gain insight into the mechanisms activated by Myc down-regulation, we have now developed an experimental model that allows modulating Myc protein expression in melanoma cells. This was achieved by originating stable melanoma cell clones expressing ecdysone-inducible c-myc antisense RNA. We show that the induction of c-myc antisense RNA in M14 melanoma cells leads to an inhibition of cell proliferation characterized by accumulation of cells in the G1 phase of the cell cycle (up to 80%) and activation of apoptosis (50%). These data are associated with an increase of p27kip1 levels and a significant reduction of the cdk2-associated kinase activity. In addition, we show that an ectopic overexpression of p27kip1 in this experimental model can enhance the apoptotic rate. Our results indicate that down-regulation of Myc protein induces a G1 arrest and activates apoptosis by increasing p27kip1 content in melanoma cells, that are known to be defective for the p16-cyclinD/cdk4-pRb G1 checkpoint. This is particularly relevant for identifying new therapeutic strategies based on the re-establishment of the apoptotic pathways in cancer cells.

Valproic acid induces neuroendocrine differentiation and UGT2B7 up-regulation in human prostate carcinoma cell line

Prostate cancer originates as an androgen-dependent hyperproliferation of the epithelial cells of the gland and it evolves in an androgen-independent, highly aggressive cancer for which no successful therapy is available to date. Neuroendocrine (NE) differentiation plays an important role in the progression of prostate cancer to an androgen-independent state with profound impact on prostate cancer (CaP) therapies. Actually, new approaches on treating advanced prostate cancer are focused on modulators of epigenetic transcriptional regulation. A new class of antitumoral agents is emerging: histone deacetylase (HDAC) inhibitors are interesting for their ability to arrest cell growth, to induce cell differentiation, and in some cases, to induce apoptosis of cancer cells. We studied the effect of valproic acid (VPA), an inhibitor of HDAC, in the human prostate androgen-dependent cancer cell line LNCaP. We observed that VPA promotes neuroendocrine-like differentiation associated with an increase in the expression of neuron-specific enolase, a decrease in prostate-specific antigen, and a down-regulation of androgen receptor protein, suggesting a modulation in the responsiveness to androgen therapy. Furthermore, selective gene expression profiling using a low-density microarray showed that VPA was able to modulate the expression of different androgen metabolism genes. We observed a down-regulation of androgen receptor coregulator (ARA24) and prostate-specific antigen, and an up-regulation of some of the UDP-glucuronosyltransferases (UGT2B11 and UGT2B7) implicated in catabolism of dihydrotestosterone (DHT) was detected. Even though UGT2B7 has only about one-tenth to one-hundredth the activity of UGT2B15 and 2B17 toward active androgens and we did not found any modulation in gene expression of these enzymes, it can be hypothesized that VPA might enhance DHT catabolism in this in vitro model and induces NE differentiation. Our data seem to raise concern about CaP treatment with VPA.

Synthesis and characterization of new transition metal complexes containing DNA intercalators of the acridine family

The coordinating ability of two DNA intercalates of the acridine family, 4-hydroxyacridine (4-OH-Acrid) and acridine orange (AO), with the transition metal ions Ni(II), Pd(II) and Pt(II), are investigated. Octahedral complexes, [Ni(4-O-Acrid)2(en)] (1), and [Ni(4-O-Acrid)2(H2O)2] (2), are obtained when the 4-OH-Acrid ligand reacts with [Ni(Cl)2(en)2] and NiCl2·6H2O, respectively. Single crystal X-ray analysis of complex 1 has shown that two acridine molecules are bonded to nickel through their nitrogen and oxygen atoms in a chelate fashion. Moreover, when AO reacts with different Pd(II) and Pt(II) precursors, new triamine complexes of the general formula cis-[M(A)n(Cl)(AO)]+X− (M = Pd(II), Pt(II); A = en, NH3; n = 1, 2; X− = BF4−, NO3−, PF6−) (3–6) are synthesized. Full characterization by IR, 1H NMR and electrospray ionization mass spectrometry in solution has demonstrated the coordination of the AO ligand to the metal ion through the endocyclic nitrogen atom. The biological activity of these new acridine orange derivatives is conducted on complex cis-[Pt(NH3)2Cl(AO)][NO3] (5). The cytotoxic activity of complex 5 is compared with that of cisplatin (cis-DDP) in DU145, A2780 and A2780-cp8 cancer cell lines and the results show that its activity is twice as effective as that of cisplatin in the two cis-DDP resistant cell lines DU145 and A2780-cp8, respectively.

Oligopeptides impairing the Myc-Max heterodimerization inhibit lung cancer cell proliferation by reducing Myc transcriptional activity.

Deregulated CMYC gene causes cell transformation and is often correlated with tumor progression and a worse clinical outcome of cancer patients. The transcription factor Myc functions by heterodimerizing with its partner, Max. As a strategy to inhibit Myc activity, we have synthesized three small peptides corresponding to segments of the leucine zipper (LZ) region of Max. The purpose of these peptides is to occupy the site of recognition between Myc and Max located in the LZ and inhibit‐specific heterodimerization between these proteins. We have used the synthesized oligopeptides in two lung cancer cell lines with different levels of Myc expression. Results demonstrate that: (i) the three peptides resulted equally effective in competing the interaction between Myc and Max in vitro; (ii) they were efficiently internalized into the cells and significantly inhibited cell growth in the cells showing the highest Myc expression; (iii) one specific peptide, only nine aminoacids long, efficiently impaired the transcriptional activity of Myc in vivo, showing a more stable interaction with this protein. Our results are relevant to the development of novel anti‐tumoral therapeutic strategies, directed to Myc‐overexpressing tumors. J. Cell. Physiol. 210: 72–80, 2007.

GSTP1*B allele increases the risk for asthma in children.

Paolo Gravina*, Federica Angelini, Marco Chianca, Alessandra Valentini, Lorenza Bellincampi, Roberta Iannini, Andrea Pierantozzi, Viviana Moschese, Elisabetta Mrak, Giorgio Federici, Loredana Chini and Sergio Bernardini 1 Department of Laboratory Medicine, Clinical Molecular Biology Laboratory, PTV, Rome, Italy 2 Department of Internal Medicine, Tor Vergata University, Rome, Italy 3 Department of Paediatrics, Tor Vergata University, Rome, Italy 4 Bambino Gesù Children’s Hospital, IRCCS, Rome, Italy

Itch gene polymorphisms in healthy population and in patients affected by rheumatoid arthritis and atopic dermatitis

Itch is a HECT-containing E3 ligase that induces proteasomal degradation of many proteins. Two targets of Itch, JunB and Notch, have been found involved in the activation of T-helper cells. It has been proposed that alterations in pathways leading to Th1 and Th2 differentiations could be involved in inflammatory diseases and in autoimmune disorders respectively. Moreover knockout mice for Itch gene displayed inflammatory immune responses and constant itching of the skin. The aim of this work was to screen the putative functional regions of Itch in order to investigate if gene polymorphisms are present in healthy population and if they are differently represented in patients affected by rheumatoid arthritis or atopic dermatitis. Genomic DNA purified from blood samples of 100 healthy volunteers, 25 atopic dermatitis, and 45 rheumatoid arthritis patients were analysed by sequencing. We found 8 substitutions in the functional regions of Itch, but we could not find significant differences between patients and healthy subjects, suggesting a critical role for Itch in the biology of the cell and that Itch could be involved in these disorders through a complex network of interactions in the proteasomal pathways.